Chapter 3 Apparent Involvement of the A2A Subtype Adenosine Receptor in the Anti-Inflammatory Interactions of CGS 21680, Cyclopentyladenosine and IS-MECA with Human Neutrophils
The wide-ranging, receptor-mediated, physiologic activities of adenosine involve interactions of this agent with at least four types of plasma membrane receptors, designated A1, A2A, A2B and A3. These vary with respect to ligand binding properties, tissue distribution and transductional mechanisms utilized in intracellular signalling (Stiles, 1992; Cronstein , 1994). Although adenosine is an important regulator of many physiologic processes, including immune and inflammatory responses, its chemotherapeutic potential is limited by an extremely short half life in vivo and by receptor promiscuity (Stiles, 1992; Cronstein, 1994). These problems have resulted in the development of synthetic agonists which are selective for the different types of AR.
The broad spectrum anti-inflammatory properties of adenosine and its analogues are well-recognized and span many different types of immune and inflammatory cells, including neutrophils (Cronstein, 1994; Hannon et a/., 1998) and eosinophils (Walker et a/. , 1997; Ezeamuzi & Philips, 1999). There is compelling evidence for the presence of A2A receptors on human neutrophils (Fredholm et a/., 1996; Varani et a/., 1998), while indirect evidence supports the existence of A1 and A3 receptors on these cells, as well as on eosinophils (Rose et a/., 1988; Bouma et a/., 1997; Fredholm, 1997; Walker et a/., 1997; Ezeamuzie & Philips, 1999). With respect to neutrophils, A1 and A2A receptors have been reported to exert opposing effects on the pro inflammatory activities of these cells . Interaction of adenosine or its analogues with A1 receptors on neutrophils has been reported to potentiate adherence to vascular endothelium and chemotaxis (Cronstein et a/., 1985; Cronstein et a/., 1992), while activation of A2A receptors results in suppression of the production of reactive oxidants by these cells (Cronstein et a/., 1985; Hannon et a/., 1998), as well as decreased expression of rs2-integrins and adherence to vascular endothelium (Cronstein et a/., 1992; Nolte et a/., 1992). Neutrophil degranulation on the other hand has been reported to be either insensitive to adenosine (Cronstein et a/., 1985), or to be inhibited by mechanisms involving both A2 and A3 receptors (Bouma et a/. , 1997). In the case of eosinophils, interaction of adenosine or its analogues with A3 receptors appears to promote down-regulation of the pro-inflammatory activities of these cells (Walker et aI. , 1997; Ezeamuzie & Philips, 1999).
Although adenosine and its analogues acting via A2A receptors suppress some of the pro-inflammatory activities of activated neutrophils, there are several aspects of this relationship , including the involvement of cAMP and the apparent insensitivity of degranulation, which require clarification. With this in mind, the current study was undertaken to identify the AR types involved in regulating the reactive oxidant generating and degranulation responses of activated human neutrophils, as well as the dependence of these anti-inflammatory activities on receptor-mediated increases in intracellular cAMP .
Materials and Methods
Adenosine receptor agonists
N6-cyclopentyladenosine (CPA, A1 R agonist), 2(4-[(2-carboxyethyl)phenyl] ethylamino)-5′-N-ethylcarboxamido adenosine – CGS 21680, A2AR agonist) and N6 _ (3-iodobenzyl)-5’N-methylcarbamoyladenosine (IB-MECA, A3R agonist) and rolipram , a selective inhibitor of type 4 phosphodiesterase, the predominant type found in human neutrophils (Torphy, 1998), were kindly provided by Dr Malcolm Johnson, GlaxoWelicome pic, Stockley Park West, London, UK. These agents were dissolved to stock concentrations of 10 mM in 0.05 N HCI (CPA and IB-MECA), 0.1 N NaOH (CGS 21680) or dimethylsulfoxide (rolipram) and diluted thereafter in indicator-free Hanks’ balanced salt solution (HBSS, pH 7.4) and used in the various assays described below at a final concentration range of 0.01-1 pM . ZM 241385, a highly selective antagonist of A2A receptors (Poucher et al’ J 1995), was purchased from Tocris Cookson Ltd , Bristol, UK and dissolved to 10mM in 0.1 N NaOH and used at concentrations of 0.1- 2.5 \-1M . Unless indicated all other chemicals and reagents were purchased from the Sigma Chemical Co.
Purified neutrophils were prepared from heparinised (5 units of preservative-free heparin/ml) venous blood of healthy adult human volunteers and separated from mononuclear leucocytes by centrifugation on Histopaque®-1077 (Sigma Diagnostics) cushions at 400 g for 25 min at room temperature. The resultant pellet was suspended in phosphate-buffered saline (PBS, 0.15 M, pH 7.4) and sedimented with 3% gelatine to remove most of the erythrocytes. After centrifugation, erythrocytes were removed by selective lysis with 0.84% ammonium chloride at 4° C for 10 min. The neutrophils, which were routinely of high purity (>90%) and viability (>95%), were resuspended to 1 x 107/ml in PBS and held on ice until used.
This was measured using a lucigenin (bis-N-methylacridinium nitrate)-enhanced chemiluminescence (LECL) method (Minkenberg & Ferber, 1984). Neutrophils (1 x 106/ml , final) were pre-incubated for 15 min in 900 ~I HBSS containing 0.2 mM lucigenin in the presence and absence of the AR agonists (0.01-1 ~M) prior to activation with the synthetic chemotactic tripeptide FMLP (1 ~M). Spontaneous and FMLP (1 ~M)-activated LECL responses were then recorded using a LKB Wallac 1251 chemiluminometer after the addition of the stimulant (100 ~I). LECL readings were integrated for 5s intervals and recorded as mV x seconds-1 (mVs-\ Additional experiments were performed to investigate the following: i) the effects of ZM 241385 (2.5 ~M) added during preincubation at 3TC, 5 min before the AR agonists on the CGS 21680, CPA and IB-MECA (1 ~M)-mediated inhibition of the LECL responses of FMLP-activated neutrophils; adenosine (1 ~M) was also included in these experiments to monitor the activity of ZM 241385, ii) the effects of low concentrations (0 .1 and 0.25 ~M) of ZM 241385 on the inhibition of FMLP-activated neutrophil superoxide production mediated by CGS 21680, CPA and IB-MECA (all at 1 ~M) and iii) the superoxide-scavenging activity of CGS 21680, CPA, IB-MECA and ZM 241385 using a cell-free hypoxanthine (1 mM)-xanthine oxidase (17 milliunits/ml) superoxide-generating system.
Neutrophil degranulation was measured according to the extent of release of the primary granule-derived protease, elastase. Neutrophils were incubated at a concentration of 2 x 106/ml in HBSS in the presence or absence of the AR agonists (0 .01-1 iJM) with and withoutZM 241385 (0.1-2 .5 iJM) for 10 min at 3rC . The stimulant FMLP (0 .1 iJM) in combination with CB (1 iJM) was then added and the reaction mixtures incubated for 10 min at 3rC. The tubes were then transferred to an ice-bath, followed by centrifugation at 400 g for 5 min to pellet the cells. The neutrophil-free supernatants were then decanted and assayed for elastase activity using a micro-modification of a standard spectrophotometric procedure (Beatty et al. , 1982). Briefly, 125 iJl of supernatant was added to 125 iJl of the elastase substrate N succinyl-L-alanyl-L-alanyl-L-alanine-p-nitroanilide, 3 mM in 0.3% dimethyl sulfoxide in 0.05 M Tris-HCI (pH 8.0). Elastase activity was assayed at a wavelength of 405 nm and the results expressed as the mean percentage of the amount of enzyme released by the corresponding FMLP/CB-activated , drug-free control systems.
Intracellular cAMP levels
Neutrophils at a concentration of 2 x 106/ml in HBSS were preincubated for 10 min at 3rC with CGS 21680, CPA or IB-MECA (1 iJM) with and without ZM 241385 (2 .5 iJM) . Following preincubation, the cells were activated with 1 iJM FMLP (stimulated cells), or an equal volume of HBSS (unstimulated cells), in a final volume of 1 ml, and the reactions terminated and the cAMP extracted by the addition of ice-cold ethanol (65% v/v) at 20 sec, 1 min, 3 min and 5 min after addition of the stimulant. The resultant precipitates were washed twice with ice-cold ethanol and the supernatants pooled and centrifuged at 2000g for 15 min at 4° C. The supernatants were then transferred to fresh tubes and evaporated at 60°C under a stream of nitrogen . The dried extracts were reconstituted in assay buffer (0.05 M acetate buffer, pH 5.8) and assayed for cAMP using the Biotrak cAMP [125 1] scintillation proximity assay system (Amersham International pic.), which is a competitive binding radioimmunoassay procedure. These results are expressed as pmoles cAMP/107 neutrophils. Because cAMP is rapidly hydrolysed in neutrophils by phosphodiesterases, these experiments were performed in the presence of 1 iJM rolipram .
Spectrofluorimetric measurement of Ca2 + fluxes
Fura-2/AM (Calbiochem Corp) , was used as the fluorescent, Ca2+-sensitive indicator for these experiments. Neutrophils (1 x 107/m l) were pre-loaded with fura-2 (2 ~M) for 30 min at 3rC in phosphate-buffered saline (PBS, 0.15 M, pH 7.4) , washed twice and resuspended in HBSS. The fura-2-loaded cells (2 x 106/ml) were then pre incubated with CPA, CGS 21680 or IB-MECA (0.01-1 ~M) at 3rC for 10 min after which they were transferred to disposable reaction cuvettes, which were maintained at 3rC in a Hitachi 650-1 OS fluorescence spectrophotometer with excitation and emission wavelengths set at 340 nm and 500 nm respectively. After a stable base line was obtained (1 min), the neutrophils were activated by addition of FMLP (1 ~M) and the subsequent increase in fura-2 fluorescence intensity monitored over a 5 min period. The final volume in each cuvette was 3 ml containing a total of 6 x 106 neutrophils. Cytoplasmic Ca2+ concentrations were calculated as described previously (Grynkiewicz et aI., 1985) .
Additional experiments were performed to investigate the effects of pre-treatment with the selective A2A receptor antagonist ZM 241385 at 2.5 ~M on CGS 21680, CPA, and IB-MECA (1 ~M)-mediated alterations in the fura-2 fluorescence responses of FMLP-activated neutrophils
Neutrophils (1 x 106ml) were preincubated for 10 min with and without CGS (1 ~M) in HBSS prior to the addition of the synthetic chemotactic tripeptide N-formyl-L methionyl-L-Ieucyl-L-phenylalanine (FMLP, 1 ~M, final) . FMLP-free control systems received an equal volume of HBSS. The final volume in each tube was 2 ml. Total and extracellular IL-8 were measured using antibody capture ELISA procedures (Roche Diagnostics Corp, Indianapolis) after 6 hours of incubation at 3rC following the addition of FMLP to the cells. TotallL-8 was measured in the Iysates of neutrophils which had been treated with 0.01 % Iysophosphatidylcholine followed by centrifugation at 300 g for 5 min to remove cell debris, while extracellular cytokines were measured in cell-free supernatants following the removal of the cells by centrifugation .
Calculations were performed by using the standards provided with the kit to prepare a six point calibration curve and a standard curve was plotted correlating the mean absorbance values of the standards (y-axis) to the analyte concentrations of the standard (x-axis). The lot-specific concentration of each standard is listed on its bottle label. Analyte concentrations were determined by locating the mean sample absorbance on the y-axis and reading from the x-axis the analyte concentration that corresponds to the specific absorbance value (h-lnterleukin-8 ELISA, cat. No 1 967 932, Boehringer Mannheim) .
The results of each series of experiments are expressed as the mean values ± SEM. Levels of statistical significance were calculated by paired Student’s t test when comparing two groups, or by analysis of variance (ANOVA) with subsequent Tukey Kramer multiple comparisons test for multiple groups . A computer-based software system (Instat II®) was used for analysis.
The effects of CGS 21680, CPA and IB-MECA on superoxide production by neutrophils activated with FMLP are shown in Figure 3.1 (page 67). Superoxide production was inhibited by CGS 21680 at all concentrations tested (0.01-1 IJM) with maximal inhibition observed at concentrations of 0.1-1 1J1’v1. IB-MECA was less effective, causing significant inhibition of superoxide production only at concentrations of 0.5 and 1 IJM, while CPA was the least effective, causing inhibition only at 1 IJM .
The effect of pre-treatment of neutrophils with ZM 241385 (2 .5 IJM) on the inhibition of the production of superoxide by FMLP-activated neutrophils mediated by 1 IJM CGS 21680, CPA and IB-MECA are shown in Table 3.1 (page 68). The A2A receptor antagonist per se slightly increased superoxide production, and also neutralized the inhibitory effects of all 3 AR agonists. ZM 241385 (2.5 IJM) also inhibited the effects of adenosine (1 IJM) on superoxide production by FMLP-activated neutrophils, with the responses of neutrophils exposed to adenosine only or to adenosine + ZM 241385 being 57 ± 4 % (p<0.05) and 109 ± 7 % of the corresponding drug-free control system respectively.
The effects of low concentrations of ZM 241385 (0.1 and 0.25 IJM) on CGS 21680- , CPA- and IB-MECA (all at 1 1J1\J1)-mediated inhibition of superoxide production by FMLP-activated neutrophils are shown in Table 3.2 (page 68). The inhibitory effects of CPA and IB-MECA on neutrophil superoxide production were completely attenuated by ZM 241385 at both concentrations used, while those of CGS 21680 were completely neutralised only at 0.25 IJM ZM 241385.
In experiments designed to evaluate the superoxide-scavenging potential of CGS 21680, CPA and IB-MECA, all 3 AR agonists at the highest concentration tested (1 IJM), as well as ZM 241385 (2.5 IJM) did not possess superoxide-scavenging properties . LECL values for the control system and for systems containing CGS 21680, CPA, IB-MECA and ZM 241385 were 1464 ± 51 , 1419 ± 159,1451 ± 75, 1412 ± 165 and 1443 ± 131 mV.s·1 respectively (results of 12 experiments).
The effects of the 3 AR agonists on elastase release from FMLP/CB-activated neutrophils are shown in Figure 3.2 (page 70). CGS 21680 and IB-MECA caused dose-related inhibition of elastase release which was evident at 0.01 IJM, while CPA exerted inhibitory effects at 1 IJM.
Table of Contents
List of Figures
List of Tables
List of Abbreviations
Chapter 1: Introduction and Literature Review
1.2 Literature Review
1.2.1 Origins of human neutrophils
1.2.2 Neutrophil granules
1.3 Neutrophil Functions
1.3.1 Extravasation and chemotaxis
1.3.2 Adherence to vascular endothelium
1.3.3 Tethering and rolling
1.3.4 Firm adhesion
1.3.5 Transendothelial migration
1.3.6 Migration of neutrophils within interstitial tissLles
1.4 Antimicrobial Mechanisms of Neutrophils
1.4.1 NADPH oxidase
1.4.2 Nitric oxide synthase
1.4.3 Cytokine production by neutrophils
1.5 Phospholipase A2-Derived Mediators of Inflammation
1.6 Calcium Fluxes and Restoration of Calcium Homeostasis in Activated Neutrophils
1.6.1 Release of calcium from stores
1.6.2 Restoration of calcium homeostasis
1.7 Anti-Inflammatory Actions of cAMP
1.7.1 Cyclic AMP and neutrophils
1.8 Neutrophil-Directed, Anti-Inflammatory Chemotherapeutic Strategies
1.8.2 Limitations of corticosteroids
1.8.3 Neutrophils and corticosteroids
1.9 Adenosine and Neutrophils
1.9.1 Adenosine effects on neutrophil function
1.9.2 Adenosine and polypeptide mediators of inflammation
1.9.3 Adenosine receptors (ARs)
Chapter 2: Effects of Dexamethasone on the Early- and Late-Activatable Pro-Inflammatory Functions of Human Neutrophils
2.2 Materials and Methods
2.2.1 Chemicals and reagents
2.2 .2 Neutrophils
2.2.3 Oxidant generation
2.2.4 Elastase release
2.2.5 Interleukin-8 production by neutrophils
2.2.6 Statistical analysis
2.3.1 Effects of dexamethasone on superoxide production by ,neutrophils
2.3.2 Effects of dexamethasone on the release of elastase by neutrophils
2.3.3 Effects of dexamethasone on the release of IL-8 from unstimulated and FMLP-activated neutrophils
Chapter 3: Apparent Involvement of the A2A Subtype Adenosine Receptor in the Anti-Inflammatory Interactions of CGS 21680, Cyclopentyladenosine and IS-MECA with Human Neutrophils
3.2 Materials and Methods
Chapter 4: Accelerated Resequestration of Cytosolic Calcium and Suppression of the Pro-Inflammatory Activities of Human Neutrophils by CGS 21680 In Vitro
4.2 Materials and Methods
Chapter 5: Concluding Comments
Chapter 6: References
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